The invention relates to a method for inspecting a sample using an assembly comprising a scanning electron microscope and a light microscope.
It is well known that electron microscopy offers nanometer-scale spatial resolution. However, when the material to be visualized is exposed to very high current densities, the imaging capabilities may be seriously limited.
On the one hand, local charging of insulating or weakly conductive materials causes imaging artefacts due to deflection of the primary imaging beam.
On the other hand, the presence of free charge carriers (electrons or possibly holes) may make the sample vulnerable to chemical reactions, such as decomposition due to bond breakage or reactions involving radical or ionized intermediates. Similarly, also the excitation of bound electrons to high energy, weakly bound states as a result of the cascade of energy dissipation reactions after electron beam irradiation, can lead to chemical modification.
Especially organic materials, such as biological material, polymers, and other soft matter, are for this reason hard to image by means of an electron microscope. However, also inorganic materials may be prone to electron-irradiation induced damage. And even for conducting materials, the decomposition of absorbed gas molecules present in the electron microscope vacuum chamber can modify the surface structure such that the imaging quality is negatively affected.
It is an object of the present invention to provide a method for imaging vulnerable and/or dynamic materials in an undamaged state or in a way that minimizes the chance for imaging artefacts or sample modification by non-invasive determination of regions of interest in-situ in the scanning electron microscope at very high spatial and temporal resolution. In addition or alternatively, it is an object of the present invention to provide a method to monitor sample damage during image scanning, and preferably to adjust the scan parameters during image acquisition based on information from the monitored sample damage.